Plant Regulatory Elements for Recombinase Autoexcision
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Solution Overview
Problem
Existing methods for removing marker genes and unwanted expression cassettes from transgenic plants are inefficient and require extensive experimentation to identify suitable promoters for site-specific recombinase expression, limiting their effectiveness across different crop species.
Innovation Solution
Development of gene regulatory elements, including meiotic promoters and 3' UTRs, operably linked to site-specific recombinases, to facilitate efficient autoexcision of marker and expression cassettes in transgenic plants, using constructs with recombinase recognition sites for precise excision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the two T-DNA transformation system is used to remove marker genes, then marker-free transgenic plants can be generated, but the efficiency is low and multiple generations are required
Solution Approach 1:
The patent incorporates the site-specific recombinase gene into the T-DNA construct before transformation, so that the recombinase is expressed in the transformed cells and can immediately excise the marker gene cassette flanked by recombination sites. This preliminary preparation of the excision mechanism eliminates the need for subsequent generations of crossing and selection, achieving marker-free plants in a single transformation event.
Solution Approach 2:
The transformed plant cell uses its own expressed site-specific recombinase to excise the marker gene cassette from its genome. The recombinase is produced by the transformed construct itself, allowing the plant to perform self-excision of unwanted sequences without requiring external intervention or additional transgenic lines.
2Manufacturing precision
If site-specific recombinase systems are used for excision, then marker gene removal can be achieved, but specificity and efficiency in excising unwanted sequences remain challenging
Solution Approach 1:
The patent uses site-specific recombinase recognition sequences (such as LoxP sites) that are specifically positioned to flank only the marker gene cassette and other unwanted sequences, while leaving desired transgene cassettes unaffected. This localized placement of recombination sites ensures precise excision of specific targets without collateral effects on other genetic elements.
Solution Approach 2:
The site-specific recombinase acts as an intermediary enzyme that catalyzes the excision of DNA sequences flanked by specific recognition sites. The recombinase binds to the LoxP sites, mediates strand exchange, and facilitates precise excision of the marker gene cassette, achieving both high specificity and efficient removal of unwanted sequences.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient and streamlined removal of marker genes and expression cassettes in transgenic plants, reducing the need for complex crossing strategies and improving genetic engineering efficiency across various crop species.
Implementation Method 1
removal of the marker gene expression cassette can be done using a two T-DNA transformation system or a site-specific recombinase system
Data Source
AI summary
Recombinant DNA molecules and constructs are provided that are useful for modulating gene expression in plants. One or more expression cassette(s) of a recombinant DNA molecule or construct may be excised from transgenic plants following transformation by the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct by expression of a recombinase enzyme encoded by the recombinant DNA molecule or construct. Such a recombinase system may be used to remove such expression cassette(s) from plants transformed with the recombinant DNA construct or vector. The recombinase transgene may be operably linked to a tissue-preferred or tissue-specific promoter for autoexcision in transformed plants without crossing to a different transgenic line expressing the recombinase. Methods for causing autoexcision of one or more expression cassette(s) in a transgenic plant, and plants and cells containing or transformed with a recombinant DNA molecule or construct of the present disclosure, are also provided.